Gao Di, Tian Teng-Hui, Yu Ke-Ying, Shao Xiao, Xue Wen, Zhao Zhi-Xuan, Deng Yue
College of Traditional Chinese Medicine, Changchun University of Chinese Medicine Changchun 130117, China.
Affiliated Hospital, Changchun University of Chinese Medicine Changchun 130021, China.
Zhongguo Zhong Yao Za Zhi. 2025 Apr;50(7):1908-1919. doi: 10.19540/j.cnki.cjcmm.20250102.501.
The purpose of this study was to clarify the effect of Huotan Jiedu Tongluo Decoction on atherosclerosis(AS) injury in ApoE(-/-) mice by regulating the ferroptosis pathway. Seventy-five ApoE(-/-) mice were randomly divided into model group, low-, medium-, and high-dose of Huotan Jiedu Tongluo Decoction groups, and evolocumab group(n=15), and 15 C57BL/6J mice were selected as the blank group. Mice in the blank group were fed with a normal diet, and those in the other groups were fed with a high-fat diet to induce AS. From the 9th week, mice in Huotan Jiedu Tongluo Decoction groups were administrated with Huotan Jiedu Tongluo Decoction at corresponding doses by gavage, and those in the blank group and the model group were given an equal volume of distilled water. Mice in the evolocumab group were treated with evolocumab 18.2 mg·kg(-1 )by subcutaneous injection every 2 weeks. After 8 weeks of continuous intervention, oil red O staining and hematoxylin-eosin(HE) staining were employed to observe the lipid deposition and plaque formation in the aortic root. Masson staining was used to evaluate the collagen content in the aortic root. The serum levels of total cholesterol(TC), triglycerides(TG), high-density lipoprotein cholesterol(HDL-C), and low-density lipoprotein cholesterol(LDL-C) were determined by biochemical kits. The levels of Fe(2+), superoxide dismutase(SOD), malondialdehyde(MDA), and glutathione(GSH) in the aorta were measured by colorimetry. The protein and mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), solute carrier family 7 member 11(SLC7A11), and acyl-CoA synthetase long chain family member 4(ACSL4) in the aorta were detected by Western blot and RT-qPCR, respectively. The expression of Nrf2, GPX4, and SLC7A11 was localized by immunofluorescence. The results showed that low-, medium-, and high-dose Huotan Jiedu Tongluo Decoction reduced the plaque formation of aortic root and increased the collagen content in AS mice. At the same time, Huotan Jiedu Tongluo Decoction improved the lipid metabolism by lowering the levels of TC, LDL-C, and TG and elevating the level of HDL-C in the serum. Huotan Jiedu Tongluo Decoction enhanced the antioxidant capacity by elevating the levels of GSH and SOD and lowering the level of MDA in the aorta and inhibiting the accumulation of Fe(2+) in the aorta. In addition, Huotan Jiedu Tongluo Decoction up-regulated the protein and mRNA levels of Nrf2, GPX4, and SLC7A11, while down-regulating the protein and mRNA levels of ACSL4. In summary, Huotan Jiedu Tongluo Decoction can effectively alleviate AS lesions in ApoE(-/-) mice by activating the Nrf2/GPX4 pathway, reducing lipid peroxidation, and inhibiting ferroptosis.
本研究旨在通过调节铁死亡途径，阐明化痰解毒通络方对载脂蛋白E基因敲除（ApoE(-/-)）小鼠动脉粥样硬化（AS）损伤的影响。将75只ApoE(-/-)小鼠随机分为模型组、化痰解毒通络方低、中、高剂量组和依洛尤单抗组（n = 15），选取15只C57BL/6J小鼠作为空白组。空白组小鼠给予正常饮食，其他组小鼠给予高脂饮食以诱导AS。从第9周起，化痰解毒通络方组小鼠按相应剂量灌胃给予化痰解毒通络方，空白组和模型组小鼠给予等体积蒸馏水。依洛尤单抗组小鼠每2周皮下注射18.2 mg·kg(-1)依洛尤单抗。连续干预8周后，采用油红O染色和苏木精-伊红（HE）染色观察主动脉根部脂质沉积和斑块形成情况。采用Masson染色评估主动脉根部胶原含量。使用生化试剂盒测定血清总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）和低密度脂蛋白胆固醇（LDL-C）水平。采用比色法测定主动脉中Fe(2+)、超氧化物歧化酶（SOD）、丙二醛（MDA）和谷胱甘肽（GSH）水平。分别通过蛋白质免疫印迹法（Western blot）和逆转录-定量聚合酶链反应（RT-qPCR）检测主动脉中核因子红细胞2相关因子2（Nrf2）、谷胱甘肽过氧化物酶4（GPX4）、溶质载体家族7成员11（SLC7A11）和酰基辅酶A合成酶长链家族成员4（ACSL4）的蛋白质和mRNA水平。通过免疫荧光法对Nrf2、GPX4和SLC7A11的表达进行定位。结果显示，化痰解毒通络方低、中、高剂量组均可减少AS小鼠主动脉根部斑块形成，增加胶原含量。同时，化痰解毒通络方可通过降低血清中TC、LDL-C和TG水平，升高HDL-C水平来改善脂质代谢。化痰解毒通络方可通过升高主动脉中GSH和SOD水平、降低MDA水平以及抑制主动脉中Fe(2+)蓄积来增强抗氧化能力。此外，化痰解毒通络方可上调Nrf2、GPX4和SLC7A11的蛋白质和mRNA水平，同时下调ACSL4的蛋白质和mRNA水平。综上所述，化痰解毒通络方可通过激活Nrf2/GPX4途径、减少脂质过氧化和抑制铁死亡，有效减轻ApoE(-/-)小鼠的AS病变。
